bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒01‒17
forty papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.
  2. Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Niemann-Pick Type C1.
  3. Conserved and Divergent Mechanisms That Control TORC1 in Yeasts and Mammals.
  4. Lysosome function in glomerular health and disease.
  5. Proteaphagy in Mammalian Cells Can Function Independent of ATG5/ATG7.
  6. Structural insights into TSC complex assembly and GAP activity on Rheb.
  7. Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept.
  8. Chaperone-mediated autophagy sustains haematopoietic stem-cell function.
  9. FV-429 induces autophagy blockage and lysosome-dependent cell death of T-cell malignancies via lysosomal dysregulation.
  10. Phosphoproteome Analysis Reveals Estrogen-ER Pathway as a Modulator of mTOR Activity Via DEPTOR.
  11. The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.
  12. Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting.
  13. Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.
  14. Repurposing Cationic Amphiphilic Drugs and Derivatives to Engage Lysosomal Cell Death in Cancer Treatment.
  15. Is SGSH heterozygosity a risk factor for early-onset neurodegenerative disease?
  16. Analysis of Brain and Cerebrospinal Fluid from Mouse Models of the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Changes in the Lysosomal Proteome.
  17. ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation.
  18. Aspartylglucosaminuria: Clinical Presentation and Potential Therapies.
  19. Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles.
  20. Chediak-Higashi syndrome: a review of the past, present, and future.
  21. Amino acid sensory complex proteins in mTORC1 and macroautophagy regulation.
  22. Lysosomal Dysfunction and Other Pathomechanisms in FTLD: Evidence from Progranulin Genetics and Biology.
  23. Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders.
  24. Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML.
  25. Reticulon-3 Promotes Endosome Maturation at ER Membrane Contact Sites.
  26. Unsaturation, curvature and charge: effects of membrane parameters on PIK3C3/VPS34-containing complexes.
  27. SESTRINs: Emerging Dynamic Stress-Sensors in Metabolic and Environmental Health.
  28. Adaptive responses to mTOR gene targeting in hematopoietic stem cells reveal a proliferative mechanism evasive to mTOR inhibition.
  29. Kidney disease genetic risk variants alter lysosomal beta-mannosidase (MANBA) expression and disease severity.
  30. Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.
  31. Elf4 regulates lysosomal biogenesis and the mTOR pathway to promote clearance of Staphylococcus aureus in macrophages.
  32. Plasma from obese children increases monocyte-endothelial adhesion and affects intracellular insulin signaling in cultured endothelial cells: potential role of mTORC1-S6K1.
  33. Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling.
  34. Inhibition of Cathepsin S Induces Mitochondrial Apoptosis in Glioblastoma Cell Lines Through Mitochondrial Stress and Autophagosome Accumulation.
  35. Modeling Brain Pathology of Niemann-Pick Disease Type C Using Patient-Derived Neurons.
  36. Profiling the Surfaceome Identifies Therapeutic Targets for Cells with Hyperactive mTORC1 Signaling.
  37. Lipid Metabolism at Membrane Contacts: Dynamics and Functions Beyond Lipid Homeostasis.
  38. Novel pharmacotherapy: NNI-362, an allosteric p70S6 kinase stimulator, reverses cognitive and neural regenerative deficits in models of aging and disease.
  39. Identification of Cathepsin D as a Plasma Biomarker for Alzheimer's Disease.
  40. AAV-Mediated GALC Gene Therapy Rescues Alpha-Synucleinopathy in the Spinal Cord of a Leukodystrophic Lysosomal Storage Disease Mouse Model.
  1. Mol Cell Proteomics. 2019 Jan;pii: S1535-9476(20)34098-6. [Epub ahead of print]18(1): 16-27
    Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.
    Tomislav Čaval, Jing Zhu, Weihua Tian, Sanne Remmelzwaal, Zhang Yang, Henrik Clausen, Albert J R Heck.
      Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases.
    Keywords:  Cellular Organelles; Glycoprotein Pathways; Glycoproteins; Glycoproteomics; Lysosomal Disorders; Lysosome; Mannose-6-phosphate; Mannose-6-phosphate Receptors; Phosphorylation; Targeted Degradation
    DOI:  https://doi.org/10.1074/mcp.RA118.000967
  2. Int J Mol Sci. 2021 Jan 12. pii: E710. [Epub ahead of print]22(2):
    Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Niemann-Pick Type C1.
    Christin Völkner, Maik Liedtke, Andreas Hermann, Moritz J Frech.
      The lysosomal storage disorders Niemann-Pick disease Type C1 (NPC1) and Type C2 (NPC2) are rare diseases caused by mutations in the NPC1 or NPC2 gene. Both NPC1 and NPC2 are proteins responsible for the exit of cholesterol from late endosomes and lysosomes (LE/LY). Consequently, mutations in one of the two proteins lead to the accumulation of unesterified cholesterol and glycosphingolipids in LE/LY, displaying a disease hallmark. A total of 95% of cases are due to a deficiency of NPC1 and only 5% are caused by NPC2 deficiency. Clinical manifestations include neurological symptoms and systemic symptoms, such as hepatosplenomegaly and pulmonary manifestations, the latter being particularly pronounced in NPC2 patients. NPC1 and NPC2 are rare diseases with the described neurovisceral clinical picture, but studies with human primary patient-derived neurons and hepatocytes are hardly feasible. Obviously, induced pluripotent stem cells (iPSCs) and their derivatives are an excellent alternative for indispensable studies with these affected cell types to study the multisystemic disease NPC1. Here, we present a review focusing on studies that have used iPSCs for disease modeling and drug discovery in NPC1 and draw a comparison to commonly used NPC1 models.
    Keywords:  NPC1; NPC2; cholesterol; iPSCs; induced pluripotent stem cells; lysosomal storage disorders; neurodegeneration; patient-specific iPSCs
    DOI:  https://doi.org/10.3390/ijms22020710
  3. Genes (Basel). 2021 Jan 12. pii: E88. [Epub ahead of print]12(1):
    Conserved and Divergent Mechanisms That Control TORC1 in Yeasts and Mammals.
    Yuichi Morozumi, Kazuhiro Shiozaki.
      Target of rapamycin complex 1 (TORC1), a serine/threonine-protein kinase complex highly conserved among eukaryotes, coordinates cellular growth and metabolism with environmental cues, including nutrients and growth factors. Aberrant TORC1 signaling is associated with cancers and various human diseases, and TORC1 also plays a key role in ageing and lifespan, urging current active research on the mechanisms of TORC1 regulation in a variety of model organisms. Identification and characterization of the RAG small GTPases as well as their regulators, many of which are highly conserved from yeast to humans, led to a series of breakthroughs in understanding the molecular bases of TORC1 regulation. Recruitment of mammalian TORC1 (mTORC1) by RAGs to lysosomal membranes is a key step for mTORC1 activation. Interestingly, the RAG GTPases in fission yeast are primarily responsible for attenuation of TORC1 activity on vacuoles, the yeast equivalent of lysosomes. In this review, we summarize our current knowledge about the functions of TORC1 regulators on yeast vacuoles, and illustrate the conserved and divergent mechanisms of TORC1 regulation between yeasts and mammals.
    Keywords:  GTPase; TOR complex 1 (TORC1); target of rapamycin (TOR); yeast
    DOI:  https://doi.org/10.3390/genes12010088
  4. Cell Tissue Res. 2021 Jan 12.
    Lysosome function in glomerular health and disease.
    Catherine Meyer-Schwesinger.
      The lysosome represents an important regulatory platform within numerous vesicle trafficking pathways including the endocytic, phagocytic, and autophagic pathways. Its ability to fuse with endosomes, phagosomes, and autophagosomes enables the lysosome to break down a wide range of both endogenous and exogenous cargo, including macromolecules, certain pathogens, and old or damaged organelles. Due to its center position in an intricate network of trafficking events, the lysosome has emerged as a central signaling node for sensing and orchestrating the cells metabolism and immune response, for inter-organelle and inter-cellular signaling and in membrane repair. This review highlights the current knowledge of general lysosome function and discusses these findings in their implication for renal glomerular cell types in health and disease including the involvement of glomerular cells in lysosomal storage diseases and the role of lysosomes in nongenetic glomerular injuries.
    Keywords:  Crescentic glomerulonephritis; Diabetic nephropathy; Focal segmental glomerulosclerosis; Glomerular endothelial cell; Glomerulus; Lysosomal storage disorder; Lysosome; Membranous nephropathy; Mesangial cell; Podocyte
    DOI:  https://doi.org/10.1007/s00441-020-03375-7
  5. Mol Cell Proteomics. 2020 Jul;pii: S1535-9476(20)34976-8. [Epub ahead of print]19(7): 1120-1131
    Proteaphagy in Mammalian Cells Can Function Independent of ATG5/ATG7.
    Tatjana Goebel, Simone Mausbach, Andreas Tuermer, Heba Eltahir, Dominic Winter, Volkmar Gieselmann, Melanie Thelen.
      The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), β-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Because these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.
    Keywords:  Proteases; autophagy; lysosome; proteasome; protein degradation; protein turnover; subcellular analysis
    DOI:  https://doi.org/10.1074/mcp.RA120.001983
  6. Nat Commun. 2021 01 12. 12(1): 339
    Structural insights into TSC complex assembly and GAP activity on Rheb.
    Huirong Yang, Zishuo Yu, Xizi Chen, Jiabei Li, Ningning Li, Jiaxuan Cheng, Ning Gao, Hai-Xin Yuan, Dan Ye, Kun-Liang Guan, Yanhui Xu.
      Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.
    DOI:  https://doi.org/10.1038/s41467-020-20522-4
  7. Mol Ther Methods Clin Dev. 2021 Mar 12. 20 191-203
    Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept.
    Jaclyn Cadaoas, Huimin Hu, Gabrielle Boyle, Elida Gomero, Rosario Mosca, Kartika Jayashankar, Mike Machado, Sean Cullen, Belle Guzman, Diantha van de Vlekkert, Ida Annunziata, Michel Vellard, Emil Kakkis, Vish Koppaka, Alessandra d'Azzo.
      Galactosialidosis is a rare lysosomal storage disease caused by a congenital defect of protective protein/cathepsin A (PPCA) and secondary deficiency of neuraminidase-1 and β-galactosidase. PPCA is a lysosomal serine carboxypeptidase that functions as a chaperone for neuraminidase-1 and β-galactosidase within a lysosomal multi-protein complex. Combined deficiency of the three enzymes leads to accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids and manifests in a systemic disease pathology with severity mostly correlating with the type of mutation(s) and age of onset of the symptoms. Here, we describe a proof-of-concept, preclinical study toward the development of enzyme replacement therapy for galactosialidosis, using a recombinant human PPCA. We show that the recombinant enzyme, taken up by patient-derived fibroblasts, restored cathepsin A, neuraminidase-1, and β-galactosidase activities. Long-term, bi-weekly injection of the recombinant enzyme in a cohort of mice with null mutation at the PPCA (CTSA) locus (PPCA -/- ), a faithful model of the disease, demonstrated a dose-dependent, systemic internalization of the enzyme by cells of various organs, including the brain. This resulted in restoration/normalization of the three enzyme activities, resolution of histopathology, and reduction of sialyloligosacchariduria. These positive results underscore the benefits of a PPCA-mediated enzyme replacement therapy for the treatment of galactosialidosis.
    Keywords:  ERT; galactosialidosis; lysosomal NEU1; lysosomal glycoproteinosis; lysosomal storage disease; lysosomes; recombinant Protective protein/cathepsin A; β-GAL
    DOI:  https://doi.org/10.1016/j.omtm.2020.11.012
  8. Nature. 2021 Jan 13.
    Chaperone-mediated autophagy sustains haematopoietic stem-cell function.
    Shuxian Dong, Qian Wang, Yun-Ruei Kao, Antonio Diaz, Inmaculada Tasset, Susmita Kaushik, Victor Thiruthuvanathan, Aliona Zintiridou, Edward Nieves, Monika Dzieciatkowska, Julie A Reisz, Evripidis Gavathiotis, Angelo D'Alessandro, Britta Will, Ana Maria Cuervo.
      The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
    DOI:  https://doi.org/10.1038/s41586-020-03129-z
  9. Cell Death Dis. 2021 Jan 13. 12(1): 80
    FV-429 induces autophagy blockage and lysosome-dependent cell death of T-cell malignancies via lysosomal dysregulation.
    Po Hu, Jubo Wang, Yingjie Qing, Hui Li, Wenzhuo Sun, Xiaoxuan Yu, Hui Hui, Qinglong Guo, Jingyan Xu.
      It is widely accepted that lysosomes are essential for cell homeostasis, and autophagy plays an important role in tumor development. Here, we found FV-429, a synthetic flavonoid compound, inhibited autophagy flux, promoted autophagosomes accumulation, and inhibited lysosomal degradation in T-cell malignancies. These effects were likely to be achieved by lysosomal dysregulation. The destructive effects of FV-429 on lysosomes resulted in blockage of lysosome-associated membrane fusion, lysosomal membrane permeabilization (LMP), and cathepsin-mediated caspase-independent cell death (CICD). Moreover, we initially investigated the effects of autophagy inhibition by FV-429 on the therapeutic efficacy of chemotherapy and found that FV-429 sensitized cancer cells to chemotherapy agents. Our findings suggest that FV-429 could be a potential novel autophagy inhibitor with notable antitumor efficacy as a single agent.
    DOI:  https://doi.org/10.1038/s41419-021-03394-4
  10. Mol Cell Proteomics. 2019 Aug;pii: S1535-9476(20)34061-5. [Epub ahead of print]18(8): 1607-1618
    Phosphoproteome Analysis Reveals Estrogen-ER Pathway as a Modulator of mTOR Activity Via DEPTOR.
    Rafael Cuesta, Marina A Gritsenko, Vladislav A Petyuk, Anil K Shukla, Chia-Feng Tsai, Tao Liu, Jason E McDermott, Marina K Holz.
      ER-positive breast tumors represent ∼70#x0025; of all breast cancer cases. Although their treatment with endocrine therapies is effective in the adjuvant or recurrent settings, the development of resistance compromises their effectiveness. The binding of estrogen to ERα, a transcription factor, triggers the regulation of the target genes (genomic pathway). Additionally, a cytoplasmic fraction of estrogen-bound ERα activates oncogenic signaling pathways such as PI3K/AKT/mTOR (nongenomic pathway). The upregulation of the estrogenic and the PI3K/AKT/mTOR signaling pathways are frequently associated with a poor outcome. To better characterize the connection between these two pathways, we performed a phosphoproteome analysis of ER-positive MCF7 breast cancer cells treated with estrogen or estrogen and the mTORC1 inhibitor rapamycin. Many proteins were identified as estrogen-regulated mTORC1 targets and among them, DEPTOR was selected for further characterization. DEPTOR binds to mTOR and inhibits the kinase activity of both mTOR complexes mTORC1 and mTORC2, but mitogen-activated mTOR promotes phosphorylation-mediated DEPTOR degradation. Although estrogen enhances the phosphorylation of DEPTOR by mTORC1, DEPTOR levels increase in estrogen-stimulated cells. We demonstrated that DEPTOR accumulation is the result of estrogen-ERα-mediated transcriptional upregulation of DEPTOR expression. Consequently, the elevated levels of DEPTOR partially counterbalance the estrogen-induced activation of mTORC1 and mTORC2. These results underscore the critical role of estrogen-ERα as a modulator of the PI3K/AKT/mTOR signaling pathway in ER-positive breast cancer cells. Additionally, these studies provide evidence supporting the use of dual PI3K/mTOR or dual mTORC1/2 inhibitors in combination with endocrine therapies as a first-line treatment option for the patients with ER-positive advanced breast cancer.
    Keywords:  Breast cancer; Gene Expression*; Phosphoproteome; Phosphorylation; Signal Transduction*
    DOI:  https://doi.org/10.1074/mcp.RA119.001506
  11. FEBS Lett. 2021 Jan 16.
    The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.
    Andrea Gubas, Christina Karantanou, Doris Popovic, Georg Tascher, Marina E Hoffmann, Anna Platzek, Nina Dawe, Ivan Dikic, Daniela S Krause, David G McEwan.
      The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 co-precipitates with, and is directly phosphorylated by, mTOR. Using a phospho-specific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.
    DOI:  https://doi.org/10.1002/1873-3468.14041
  12. Autophagy. 2021 Jan 14.
    Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting.
    Merve Kacal, Boxi Zhang, Yuqing Hao, Erik Norberg, Helin Vakifahmetoglu-Norberg.
      Autophagic pathways are regulated mechanisms that play important roles in lysosome-mediated cellular degradation. Yet, the contribution of different autophagic pathways in lysosomal targeting, and characterization of the extent and specificity in their degradome remains largely uncharacterized. By undertaking a multiplex quantitative mass spectrometry approach, we have previously analyzed the lysosomal proteome during chaperone-mediated autophagy (CMA)-stimulated conditions in cancer cells. Here, we have extended our multiplex quantitative mass spectrometry and bioinformatics analysis on the proteome from isolated lysosomes to gain a comprehensive view of the temporal enriched lysosomal content upon non-macroautophagy-activated conditions. In parallel, we described the functional dependency of LAMP2A on, and to what degree the presence of KFERQ-like motifs in proteins influences, their lysosomal targeting. These findings establish a framework for a better understanding of the degradome mediated by autophagic pathways beyond macroautophagy, and present characterization of the impact of LAMP2A in lysosomal targeting in cancer cells.
    Keywords:  Autophagy; cancer; chaperone-mediated autophagy; lysosome; proteomics
    DOI:  https://doi.org/10.1080/15548627.2021.1876343
  13. Nat Commun. 2021 01 12. 12(1): 290
    Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.
    Jung-Hwan Kim, Tsutomu Matsubara, Jaekwon Lee, Cristina Fenollar-Ferrer, Kyungreem Han, Donghwan Kim, Shang Jia, Christopher J Chang, Heejung Yang, Tomokazu Nagano, Kristopher W Krausz, Sun-Hee Yim, Frank J Gonzalez.
      The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.
    DOI:  https://doi.org/10.1038/s41467-020-20461-0
  14. Front Oncol. 2020 ;10 605361
    Repurposing Cationic Amphiphilic Drugs and Derivatives to Engage Lysosomal Cell Death in Cancer Treatment.
    Michelle Hu, Kermit L Carraway.
      A major confounding issue in the successful treatment of cancer is the existence of tumor cell populations that resist therapeutic agents and regimens. While tremendous effort has gone into understanding the biochemical mechanisms underlying resistance to each traditional and targeted therapeutic, a broader approach to the problem may emerge from the recognition that existing anti-cancer agents elicit their cytotoxic effects almost exclusively through apoptosis. Considering the myriad mechanisms cancer cells employ to subvert apoptotic death, an attractive alternative approach would leverage programmed necrotic mechanisms to side-step therapeutic resistance to apoptosis-inducing agents. Lysosomal cell death (LCD) is a programmed necrotic cell death mechanism that is engaged upon the compromise of the limiting membrane of the lysosome, a process called lysosomal membrane permeabilization (LMP). The release of lysosomal components into the cytosol upon LMP triggers biochemical cascades that lead to plasma membrane rupture and necrotic cell death. Interestingly, the process of cellular transformation appears to render the limiting lysosomal membranes of tumor cells more fragile than non-transformed cells, offering a potential therapeutic window for drug development. Here we outline the concepts of LMP and LCD, and discuss strategies for the development of agents to engage these processes. Importantly, the potential exists for existing cationic amphiphilic drugs such as antidepressants, antibiotics, antiarrhythmics, and diuretics to be repurposed to engage LCD within therapy-resistant tumor cell populations.
    Keywords:  cancer treatment; cationic amphiphilic drugs; lysosomal cell death; lysosomal membrane permeabilization; necrosis; therapeutic repurposing; therapeutic resistance; therapeutic targeting
    DOI:  https://doi.org/10.3389/fonc.2020.605361
  15. J Inherit Metab Dis. 2021 Jan 09.
    Is SGSH heterozygosity a risk factor for early-onset neurodegenerative disease?
    Meghan L Douglass, Helen Beard, Andrew Shoubridge, Nazzmer Nazri, Barbara King, Paul J Trim, Stephen K Duplock, Marten F Snel, John J Hopwood, Kim M Hemsley.
      Lysosomal dysfunction may be an important factor in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). Heterozygous mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) have been found in PD patients, and some but not all mutations in other lysosomal enzyme genes for example, NPC1 and MCOLN1 have been associated with PD. We have examined the behaviour and brain structure of mice carrying a D31N mutation in the sulphamidase (Sgsh) gene which encodes a lysosomal sulphatase. Female heterozygotes and wildtype mice aged 12-, 15-, 18- and 21-months of age underwent motor phenotyping and the brain was comprehensively evaluated for disease-associated lesions. Heterozygous mice exhibited impaired performance in the negative geotaxis test when compared with wildtype mice. Whilst the brain of Sgsh heterozygotes aged up to 21-months did not exhibit any of the gross features of PD, Alzheimer's disease or the neurodegenerative lysosomal storage disorders for example, loss of striatal dopamine, reduced GBA activity, α-synuclein-positive inclusions, perturbation of lipid synthesis, or cerebellar Purkinje cell drop-out, we noted discrete structural aberrations in the dendritic tree of cortical pyramidal neurons in 21-month old animals. The overt disease lesions and resultant phenotypic changes previously described in individuals with heterozygous mutations in lysosomal enzyme genes such as glucocerebrosidase may be enzyme dependent. By better understanding why deficiency in, or mutant forms of some but not all lysosomal proteins leads to heightened risk or earlier onset of classical neurodegenerative disorders, novel disease-causing mechanisms may be identified. This article is protected by copyright. All rights reserved.
    Keywords:  Golgi-Cox; dendritic tree; heterozygote; lysosomal; motor cortex; negative geotaxis
    DOI:  https://doi.org/10.1002/jimd.12359
  16. Mol Cell Proteomics. 2019 Nov;pii: S1535-9476(20)31762-X. [Epub ahead of print]18(11): 2244-2261
    Analysis of Brain and Cerebrospinal Fluid from Mouse Models of the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Changes in the Lysosomal Proteome.
    David E Sleat, Jennifer A Wiseman, Mukarram El-Banna, Haiyan Zheng, Caifeng Zhao, Amenah Soherwardy, Dirk F Moore, Peter Lobel.
      Treatments are emerging for the neuronal ceroid lipofuscinoses (NCLs), a group of similar but genetically distinct lysosomal storage diseases. Clinical ratings scales measure long-term disease progression and response to treatment but clinically useful biomarkers have yet to be identified in these diseases. We have conducted proteomic analyses of brain and cerebrospinal fluid (CSF) from mouse models of the most frequently diagnosed NCL diseases: CLN1 (infantile NCL), CLN2 (classical late infantile NCL) and CLN3 (juvenile NCL). Samples were obtained at different stages of disease progression and proteins quantified using isobaric labeling. In total, 8303 and 4905 proteins were identified from brain and CSF, respectively. We also conduced label-free analyses of brain proteins that contained the mannose 6-phosphate lysosomal targeting modification. In general, we detect few changes at presymptomatic timepoints but later in disease, we detect multiple proteins whose expression is significantly altered in both brain and CSF of CLN1 and CLN2 animals. Many of these proteins are lysosomal in origin or are markers of neuroinflammation, potentially providing clues to underlying pathogenesis and providing promising candidates for further validation.
    Keywords:  Mouse models; biomarker: prognostic; cerebrospinal fluid; glycoproteins*; inflammatory response; lysosome; mass spectrometry; neurodegenerative diseases; neuronal ceroid lipofuscinosis
    DOI:  https://doi.org/10.1074/mcp.RA119.001587
  17. Dev Cell. 2020 Dec 16. pii: S1534-5807(20)31016-9. [Epub ahead of print]
    ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation.
    Guangyan Miao, Hongyu Zhao, Yan Li, Mingming Ji, Yong Chen, Yi Shi, Yuhai Bi, Peihui Wang, Hong Zhang.
      Autophagy acts as a cellular surveillance mechanism to combat invading pathogens. Viruses have evolved various strategies to block autophagy and even subvert it for their replication and release. Here, we demonstrated that ORF3a of the COVID-19 virus SARS-CoV-2 inhibits autophagy activity by blocking fusion of autophagosomes/amphisomes with lysosomes. The late endosome-localized ORF3a directly interacts with and sequestrates the homotypic fusion and protein sorting (HOPS) component VPS39, thereby preventing HOPS complex from interacting with the autophagosomal SNARE protein STX17. This blocks assembly of the STX17-SNAP29-VAMP8 SNARE complex, which mediates autophagosome/amphisome fusion with lysosomes. Expression of ORF3a also damages lysosomes and impairs their function. SARS-CoV-2 virus infection blocks autophagy, resulting in accumulation of autophagosomes/amphisomes, and causes late endosomal sequestration of VPS39. Surprisingly, ORF3a from the SARS virus SARS-CoV fails to interact with HOPS or block autophagy. Our study reveals a mechanism by which SARS-CoV-2 evades lysosomal destruction and provides insights for developing new strategies to treat COVID-19.
    Keywords:  COVID-19; DMV; HOPS; ORF3a; SARS-CoV-2; SNARE; autophagy
    DOI:  https://doi.org/10.1016/j.devcel.2020.12.010
  18. J Child Neurol. 2021 Jan 13. 883073820980904
    Aspartylglucosaminuria: Clinical Presentation and Potential Therapies.
    Kimberly Goodspeed, Cynthia Feng, Minna Laine, Troy C Lund.
      Aspartylglucosaminuria (AGU) is a recessively inherited neurodegenerative lysosomal storage disease characterized by progressive intellectual disability, skeletal abnormalities, connective tissue overgrowth, gait disturbance, and seizures followed by premature death. AGU is caused by pathogenic variants in the aspartylglucosaminidase (AGA) gene, leading to glycoasparagine accumulation and cellular dysfunction. Although more prevalent in the Finnish population, more than 30 AGA variants have been identified worldwide. Owing to its rarity, AGU may be largely underdiagnosed. Recognition of the following early clinical features may aid in AGU diagnosis: developmental delays, hyperactivity, early growth spurt, inguinal and abdominal hernias, clumsiness, characteristic facial features, recurring upper respiratory and ear infections, tonsillectomy, multiple sets of tympanostomy tube placement, and sleep problems. Although no curative therapies currently exist, early diagnosis may provide benefit through the provision of anticipatory guidance, management of expectations, early interventions, and prophylaxis; it will also be crucial for increased clinical benefits of future AGU disease-modifying therapies.
    Keywords:  adolescents; children; genetics; intellectual disability; lysosomal disease; mutation; pediatric
    DOI:  https://doi.org/10.1177/0883073820980904
  19. Chem Phys Lipids. 2021 Jan 07. pii: S0009-3084(20)30178-X. [Epub ahead of print] 105047
    Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles.
    Alice Dupont Juhl, Frederik W Lund, Maria Louise V Jensen, Maria Szomek, Christian W Heegaard, Peter Guttmann, Stephan Werner, James McNally, Gerd Schneider, Sergey Kapishnikov, Daniel Wüstner.
      The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2's known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.
    Keywords:  Niemann-Pick type C disease; cholesterol efflux; extracellular vesicles; fluorescence; image analysis; soft X-ray tomography
    DOI:  https://doi.org/10.1016/j.chemphyslip.2020.105047
  20. Drug Discov Today Dis Models. 2020 ;31 31-36
    Chediak-Higashi syndrome: a review of the past, present, and future.
    Prashant Sharma, Elena-Raluca Nicoli, Jenny Serra-Vinardell, Marie Morimoto, Camilo Toro, May Christine V Malicdan, Wendy J Introne.
      Since the initial description of Chediak-Higashi syndrome (CHS), over 75 years ago, several studies have been conducted to underscore the role of the lysosomal trafficking regulator (LYST) gene in the pathogenesis of disease. CHS is a rare autosomal recessive disorder, which is caused by biallelic mutations in the highly conserved LYST gene. The disease is characterized by partial oculocutaneous albinism, prolonged bleeding, immune and neurologic dysfunction, and risk for the development of hemophagocytic lympohistiocytosis (HLH). The presence of giant secretory granules in leukocytes is the classical diagnostic feature, which distinguishes CHS from closely related Griscelli and Hermansky-Pudlak syndromes. While the exact mechanism of the formation of the giant granules in CHS patients is not understood, dysregulation of LYST function in regulating lysosomal biogenesis has been proposed to play a role. In this review, we discuss the clinical characteristics of the disease and highlight the functional consequences of enlarged lysosomes and lysosome-related organelles (LROs) in CHS.
    Keywords:  Chediak-Higashi syndrome; LYST; Lysosome-related organelles; Lysosomes
    DOI:  https://doi.org/10.1016/j.ddmod.2019.10.008
  21. Matrix Biol. 2021 Jan 08. pii: S0945-053X(21)00001-9. [Epub ahead of print]
    Amino acid sensory complex proteins in mTORC1 and macroautophagy regulation.
    Marcel Seibert, Nina Kurrle, Frank Schnütgen, Hubert Serve.
      Autophagy is the highly conserved catabolic process, which enables the survival of a cell under unfavorable environmental conditions. In a constantly changing environment, cells must be capable of dynamically oscillating between anabolism and catabolism in order to maintain cellular homeostasis. In this context, the activity of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) is of major importance. As a central signaling node, it directly controls the process of macroautophagy and thus cellular metabolism. Thereby, the control of mTORC1 is equally crucial as the regulation of cellular homeostasis itself, whereby particular importance is attributed to amino acid sensory proteins. In this review, we describe the recent findings of macroautophagy and mTORC1 regulation by upstream amino acid stimuli in different subcellular localizations. We highlight in detail which proteins of the sensor complexes play a specific role in this regulation and point out additional non-canonical functions, e.g. in the regulation of macroautophagy, which have received little attention so far.
    DOI:  https://doi.org/10.1016/j.matbio.2021.01.001
  22. Adv Exp Med Biol. 2021 ;1281 219-242
    Lysosomal Dysfunction and Other Pathomechanisms in FTLD: Evidence from Progranulin Genetics and Biology.
    Xiaolai Zhou, Thomas Kukar, Rosa Rademakers.
      It has been more than a decade since heterozygous loss-of-function mutations in the progranulin gene (GRN) were first identified as an important genetic cause of frontotemporal lobar degeneration (FTLD). Due to the highly diverse biological functions of the progranulin (PGRN) protein, encoded by GRN, multiple possible disease mechanisms have been proposed. Early work focused on the neurotrophic properties of PGRN and its role in the inflammatory response. However, since the discovery of homozygous GRN mutations in patients with a lysosomal storage disorder, investigation into the possible roles of PGRN and its proteolytic cleavage products granulins, in lysosomal function and dysfunction, has taken center stage. In this chapter, we summarize the GRN mutational spectrum and its associated phenotypes followed by an in-depth discussion on the possible disease mechanisms implicated in FTLD-GRN. We conclude with key outstanding questions which urgently require answers to ensure safe and successful therapy development for GRN mutation carriers.
    Keywords:  Familial frontotemporal dementia; Genetic modifier; Granulins; Inflammation; Lysosomal dysfunction; Lysosome; Neurotrophic factor; Progranulin; TMEM106B
    DOI:  https://doi.org/10.1007/978-3-030-51140-1_14
  23. Science. 2021 01 15. 371(6526): 265-270
    Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders.
    Yoshikazu Johmura, Takehiro Yamanaka, Satotaka Omori, Teh-Wei Wang, Yuki Sugiura, Masaki Matsumoto, Narumi Suzuki, Soichiro Kumamoto, Kiyoshi Yamaguchi, Seira Hatakeyama, Tomoyo Takami, Rui Yamaguchi, Eigo Shimizu, Kazutaka Ikeda, Nobuyuki Okahashi, Ryuta Mikawa, Makoto Suematsu, Makoto Arita, Masataka Sugimoto, Keiichi I Nakayama, Yoichi Furukawa, Seiya Imoto, Makoto Nakanishi.
      Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, we identified glutaminase 1 (GLS1) as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.
    DOI:  https://doi.org/10.1126/science.abb5916
  24. Nat Commun. 2021 01 11. 12(1): 245
    Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML.
    Toshihiko Oki, Francois Mercier, Hiroki Kato, Yookyung Jung, Thomas O McDonald, Joel A Spencer, Michael C Mazzola, Nick van Gastel, Charles P Lin, Franziska Michor, Toshio Kitamura, David T Scadden.
      Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.
    DOI:  https://doi.org/10.1038/s41467-020-20491-8
  25. Dev Cell. 2021 Jan 11. pii: S1534-5807(20)31020-0. [Epub ahead of print]56(1): 52-66.e7
    Reticulon-3 Promotes Endosome Maturation at ER Membrane Contact Sites.
    Haoxi Wu, Gia K Voeltz.
      ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.
    Keywords:  endoplasmic reticulum; endosome maturation; membrane contact sites
    DOI:  https://doi.org/10.1016/j.devcel.2020.12.014
  26. Autophagy. 2021 Jan 15. 1-3
    Unsaturation, curvature and charge: effects of membrane parameters on PIK3C3/VPS34-containing complexes.
    Yohei Ohashi, Shirley Tremel, Roger L Williams.
      Phosphatidylinositol-3-phosphate (PtdIns3P) is essential for generating autophagosomes and regulating endocytic trafficking. Recently, we have shown that the activities of human PIK3C3/VPS34-containing complexes I and II, which synthesize PtdIns3P, are greatly affected by three membrane physicochemical parameters: lipid unsaturation, membrane curvature, and negative charge. Both complexes are more active on membranes composed of unsaturated lipids than saturated lipids, and high membrane curvature can compensate for the negative effect of high lipid saturation. Negatively charged phosphatidylserine (PS) activates the complexes, as well as PIK3C3/VPS34 alone. The kinase activity of complex I depends critically on the ATG14 BATS domain, whereas complex II relies on the BECN1 BARA domain. Our findings highlight the importance of the membrane character as sensed by the unique membrane binding motifs/domain of the complexes for regulating PIK3C3/VPS34 activity.
    Keywords:  BARA; bats; curvature; electrostatics; lipid; packing; pik3c3/VPS34; ptdins3p; unsaturation
    DOI:  https://doi.org/10.1080/15548627.2021.1872190
  27. Front Cell Dev Biol. 2020 ;8 603421
    SESTRINs: Emerging Dynamic Stress-Sensors in Metabolic and Environmental Health.
    Seung-Hyun Ro, Julianne Fay, Cesar I Cyuzuzo, Yura Jang, Naeun Lee, Hyun-Seob Song, Edward N Harris.
      Proper timely management of various external and internal stresses is critical for metabolic and redox homeostasis in mammals. In particular, dysregulation of mechanistic target of rapamycin complex (mTORC) triggered from metabolic stress and accumulation of reactive oxygen species (ROS) generated from environmental and genotoxic stress are well-known culprits leading to chronic metabolic disease conditions in humans. Sestrins are one of the metabolic and environmental stress-responsive groups of proteins, which solely have the ability to regulate both mTORC activity and ROS levels in cells, tissues and organs. While Sestrins are originally reported as one of several p53 target genes, recent studies have further delineated the roles of this group of stress-sensing proteins in the regulation of insulin sensitivity, glucose and fat metabolism, and redox-function in metabolic disease and aging. In this review, we discuss recent studies that investigated and manipulated Sestrins-mediated stress signaling pathways in metabolic and environmental health. Sestrins as an emerging dynamic group of stress-sensor proteins are drawing a spotlight as a preventive or therapeutic mechanism in both metabolic stress-associated pathologies and aging processes at the same time.
    Keywords:  ROS; Sestrins; aging; cancer; environmental stress; mTORC; metabolic disease; obesity/inflammation
    DOI:  https://doi.org/10.3389/fcell.2020.603421
  28. Proc Natl Acad Sci U S A. 2021 Jan 05. pii: e2020102118. [Epub ahead of print]118(1):
    Adaptive responses to mTOR gene targeting in hematopoietic stem cells reveal a proliferative mechanism evasive to mTOR inhibition.
    Cuiqing Fan, Chuntao Zhao, Feng Zhang, Meenu Kesarwani, Zhaowei Tu, Xiongwei Cai, Ashley Kuenzi Davis, Lingli Xu, Cindy L Hochstetler, Xiaoyi Chen, Fukun Guo, Gang Huang, Mohammad Azam, Weidong Tian, Q Richard Lu, Yi Zheng.
      The mechanistic target of rapamycin (mTOR) is a central regulator of cell growth and an attractive anticancer target that integrates diverse signals to control cell proliferation. Previous studies using mTOR inhibitors have shown that mTOR targeting suppresses gene expression and cell proliferation. To date, however, mTOR-targeted therapies in cancer have seen limited efficacy, and one key issue is related to the development of evasive resistance. In this manuscript, through the use of a gene targeting mouse model, we have found that inducible deletion of mTOR in hematopoietic stem cells (HSCs) results in a loss of quiescence and increased proliferation. Adaptive to the mTOR loss, mTOR -/- HSCs increase chromatin accessibility and activate global gene expression, contrary to the effects of short-term inhibition by mTOR inhibitors. Mechanistically, such genomic changes are due to a rewiring and adaptive activation of the ERK/MNK/eIF4E signaling pathway that enhances the protein translation of RNA polymerase II, which in turn leads to increased c-Myc gene expression, allowing the HSCs to thrive despite the loss of a functional mTOR pathway. This adaptive mechanism can also be utilized by leukemia cells undergoing long-term mTOR inhibitor treatment to confer resistance to mTOR drug targeting. The resistance can be counteracted by MNK, CDK9, or c-Myc inhibition. These results provide insights into the physiological role of mTOR in mammalian stem cell regulation and implicate a mechanism of evasive resistance in the context of mTOR targeting.
    Keywords:  adaptive compensation; drug resistance; hematopoietic stem cells; leukemia; mTOR
    DOI:  https://doi.org/10.1073/pnas.2020102118
  29. Sci Transl Med. 2021 Jan 13. pii: eaaz1458. [Epub ahead of print]13(576):
    Kidney disease genetic risk variants alter lysosomal beta-mannosidase (MANBA) expression and disease severity.
    Xiangchen Gu, Hongliu Yang, Xin Sheng, Yi-An Ko, Chengxiang Qiu, Jihwan Park, Shizheng Huang, Rachel Kember, Renae L Judy, Joseph Park, Scott M Damrauer, Girish Nadkarni, Ruth J F Loos, Vy Thi Ha My, Kumardeep Chaudhary, Erwin P Bottinger, Ishan Paranjpe, Aparna Saha, Christopher Brown, Shreeram Akilesh, Adriana M Hung, Matthew Palmer, Aris Baras, John D Overton, Jeffrey Reid, Marylyn Ritchie, Daniel J Rader, Katalin Susztak.
      More than 800 million people in the world suffer from chronic kidney disease (CKD). Genome-wide association studies (GWAS) have identified hundreds of loci where genetic variants are associated with kidney function; however, causal genes and pathways for CKD remain unknown. Here, we performed integration of kidney function GWAS and human kidney-specific expression quantitative trait analysis and identified that the expression of beta-mannosidase (MANBA) was lower in kidneys of subjects with CKD risk genotype. We also show an increased incidence of renal failure in subjects with rare heterozygous loss-of-function coding variants in MANBA using phenome-wide association analysis of 40,963 subjects with exome sequencing data. MANBA is a lysosomal gene highly expressed in kidney tubule cells. Deep phenotyping revealed structural and functional lysosomal alterations in human kidneys from subjects with CKD risk alleles and mice with genetic deletion of Manba Manba heterozygous and knockout mice developed more severe kidney fibrosis when subjected to toxic injury induced by cisplatin or folic acid. Manba loss altered multiple pathways, including endocytosis and autophagy. In the absence of Manba, toxic acute tubule injury induced inflammasome activation and fibrosis. Together, these results illustrate the convergence of common noncoding and rare coding variants in MANBA in kidney disease development and demonstrate the role of the endolysosomal system in kidney disease development.
    DOI:  https://doi.org/10.1126/scitranslmed.aaz1458
  30. Mol Cell Proteomics. 2019 Jul;pii: S1535-9476(20)31543-7. [Epub ahead of print]18(7): 1330-1344
    Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.
    Tommy Weiss-Sadan, Gal Itzhak, Farnusch Kaschani, Zhanru Yu, Mohamed Mahameed, Adi Anaki, Yael Ben-Nun, Emmanuelle Merquiol, Boaz Tirosh, Benedikt Kessler, Markus Kaiser, Galia Blum.
      Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.
    Keywords:  Cathepsin L; Gene Expression*; Glycolysis; Metabolomics; Pathway Analysis; Proliferation; Proteases*; Proteolysis*
    DOI:  https://doi.org/10.1074/mcp.RA119.001392
  31. FEBS Lett. 2021 Jan 10.
    Elf4 regulates lysosomal biogenesis and the mTOR pathway to promote clearance of Staphylococcus aureus in macrophages.
    Yanhua Kang, Tingyue Wu, Yan He, Yunfan He, Dongjiu Zhao.
      Staphylococcus aureus (S. aureus) is a major cause of infectious disease. Macrophages can directly destroy most of the invading bacteria through the phagolysosomal pathway. E74-like factor 4 (Elf4) is one of the important transcription factors that controls diverse pathogens, but the role of Elf4 in macrophage-mediated S. aureus eradication is unknown. Our data show that Elf4 is induced by S. aureus in macrophages. Elevated expression of Elf4 results in decreased bacterial load and inflammatory responses during S. aureus infection in vivo and in vitro. Elf4-overexpressed macrophages have decreased mTOR activity and increased lysosomal mass. Collectively, these results suggest that S. aureus induces Elf4 expression, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.
    Keywords:   Staphylococcus aureus ; Elf4; lysosomal biogenesis; mTOR; macrophages
    DOI:  https://doi.org/10.1002/1873-3468.14037
  32. Biochim Biophys Acta Mol Basis Dis. 2021 Jan 07. pii: S0925-4439(21)00009-0. [Epub ahead of print] 166076
    Plasma from obese children increases monocyte-endothelial adhesion and affects intracellular insulin signaling in cultured endothelial cells: potential role of mTORC1-S6K1.
    Nadia Di Pietrantonio, Carola Palmerini, Caterina Pipino, Maria Pompea Antonia Baldassarre, Giuseppina Bologna, Angelika Mohn, Cosimo Giannini, Paola Lanuti, Francesco Chiarelli, Assunta Pandolfi, Natalia Di Pietro.
      Childhood obesity is characterized by the loss of vascular insulin sensitivity along with altered oxidant-antioxidant state and chronic inflammation, which play a key role in the onset of endothelial dysfunction. We previously demonstrated a reduced insulin-stimulated Nitric Oxide (NO) bioavailability in Human Umbilical Vein Endothelial cells (HUVECs) cultured with plasma from obese pre-pubertal children (OB) compared to those cultured with plasma of normal-weight children (CTRL). However, mechanisms underlying endothelial dysfunction in childhood obesity remains poorly understood. Hence, the present study aimed to better investigate these mechanisms, also considering a potential involvement of mammalian Target Of Rapamycin Complex1 (mTORC1)-ribosomal protein S6 Kinase beta1 (S6K1) pathway. OB-children (N = 32, age: 9.2 ± 1.7; BMI z-score: 2.72 ± 0.31) had higher fasting insulin levels and increased HOMA-IR than CTRL-children (N = 32, age: 8.8 ± 1.2; BMI z-score: 0.33 ± 0.75). In vitro, HUVECs exposed to OB-plasma exhibited significant increase in Reactive Oxygen Species (ROS) levels, higher vascular and intercellular adhesion molecules exposure, together with increased monocytes-endothelial interaction. This was associated with unbalanced pro- and anti-atherogenic endothelial insulin stimulated signaling pathways, as measured by increased Mitogen Activated Protein Kinase (MAPK) and decreased Insulin Receptor Substrate-1 (IRS-1)/protein kinase B (Akt)/ endothelial NO Synthase (eNOS) phosphorylation levels, together with augmented S6K1 activation. Interestingly, inhibition of mTORC1-S6K1 pathway using rapamycin significantly restored the IRS-1/Akt/eNOS activation, suggesting a feedback regulation of IRS-1/Akt signal through S6K1. Overall, our in vitro data shed light on new mechanisms underlying the onset of endothelial dysfunction in childhood obesity.
    Keywords:  Childhood obesity; endothelial dysfunction; insulin resistance; monocytes adhesion; oxidative stress; rapamycin
    DOI:  https://doi.org/10.1016/j.bbadis.2021.166076
  33. Mol Cell Proteomics. 2020 Jul;pii: S1535-9476(20)34975-6. [Epub ahead of print]19(7): 1104-1119
    Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling.
    Samuel W Entwisle, Camila Martinez Calejman, Anthony S Valente, Robert T Lawrence, Chien-Min Hung, David A Guertin, Judit Villén.
      Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.
    Keywords:  Phosphoproteome; adipocytes; insulin signaling; mTOR; phosphorylation; signal transduction; signaling circuits; targeted mass spectrometry
    DOI:  https://doi.org/10.1074/mcp.RA120.001946
  34. Front Oncol. 2020 ;10 516746
    Inhibition of Cathepsin S Induces Mitochondrial Apoptosis in Glioblastoma Cell Lines Through Mitochondrial Stress and Autophagosome Accumulation.
    Maoxing Fei, Li Zhang, Handong Wang, Yihao Zhu, Wenhao Niu, Ting Tang, Yanling Han.
      Cathepsin S (CTSS), a lysosomal cysteine protease, is overexpressed in various cancers, including glioblastoma (GB). A high level of CTSS is associated with tumor progression and poor outcome in GB. However, the underlying mechanisms of its role in the biological characteristics of G5B remain to be elucidated. Here, we uncovered a potential role of CTSS in the lysosomes and mitochondria of GB cells (GBCs). Downregulation of CTSS in GBCs could increase the expression of autophagy-related proteins; however, there was no significant change in p62, suggesting autophagy blockade. Moreover, inhibition of CTSS increased the expression of mitochondrial calcium uniporter (MCU) and enhanced mitochondrial Ca2+ uptake ability, causing mitochondrial Ca2+ overload, the generation of copious reactive oxygen species (ROS) and eventual mitochondrial apoptosis. Additionally, elevated damage to mitochondria exacerbated the burden of autophagy. Finally, we found that silence of MCU could alleviate the inhibition of CTSS-induced autophagosome accumulation and mitochondrial stress. Collectively, these results demonstrate that CTSS plays an important role in the process of autophagic flux and mitochondrial functions in GBCs.
    Keywords:  autophagy; cathepsin S; glioblastoma; mitochondrial calcium uniporter; mitophagy
    DOI:  https://doi.org/10.3389/fonc.2020.516746
  35. Mov Disord. 2021 Jan 13.
    Modeling Brain Pathology of Niemann-Pick Disease Type C Using Patient-Derived Neurons.
    Lena F Burbulla, Jessica M Mc Donald, Clarissa Valdez, Fanding Gao, Eileen H Bigio, Dimitri Krainc.
      BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal-recessive lysosomal storage disease that is also associated with progressive neurodegeneration. NPC shares many pathological features with Alzheimer's disease, including neurofibrillary tangles, axonal spheroids, β-amyloid deposition, and dystrophic neurites. Here, we examined if these pathological features could be detected in induced pluripotent stem cell (iPSC)-derived neurons from NPC patients.METHODS: Brain tissues from 8 NPC patients and 5 controls were analyzed for histopathological and biochemical markers of pathology. To model disease in culture, iPSCs from NPC patients and controls were differentiated into cortical neurons.
    RESULTS: We found hyperphosphorylated tau, altered processing of amyloid precursor protein, and increased Aβ42 in NPC postmortem brains and in iPSC-derived cortical neurons from NPC patients.
    CONCLUSION: Our findings demonstrated that the main pathogenic phenotypes typically found in NPC brains were also observed in patient-derived neurons, providing a useful model for further mechanistic and therapeutic studies of NPC. © 2021 International Parkinson and Movement Disorder Society.
    Keywords:  Niemann-Pick disease; iPSC; disease modeling; brain pathology
    DOI:  https://doi.org/10.1002/mds.28463
  36. Mol Cell Proteomics. 2020 Feb;pii: S1535-9476(20)35080-5. [Epub ahead of print]19(2): 294-307
    Profiling the Surfaceome Identifies Therapeutic Targets for Cells with Hyperactive mTORC1 Signaling.
    Junnian Wei, Kevin Leung, Charles Truillet, Davide Ruggero, James A Wells, Michael J Evans.
      Aberrantly high mTORC1 signaling is a known driver of many cancers and human disorders, yet pharmacological inhibition of mTORC1 rarely confers durable clinical responses. To explore alternative therapeutic strategies, herein we conducted a proteomics survey to identify cell surface proteins upregulated by mTORC1. A comparison of the surfaceome from Tsc1-/-versus Tsc1+/+ mouse embryonic fibroblasts revealed 59 proteins predicted to be significantly overexpressed in Tsc1-/- cells. Further validation of the data in multiple mouse and human cell lines showed that mTORC1 signaling most dramatically induced the expression of the proteases neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). Functional studies showed that constitutive mTORC1 signaling sensitized cells to genetic ablation of NEP and APN, as well as the biochemical inhibition of APN. In summary, these data show that mTORC1 signaling plays a significant role in the constitution of the surfaceome, which in turn may present novel therapeutic strategies.
    Keywords:  SILAC; cancer biology; cancer therapeutics; cell biology; drug targets; membranes; mouse models
    DOI:  https://doi.org/10.1074/mcp.RA119.001785
  37. Front Cell Dev Biol. 2020 ;8 615856
    Lipid Metabolism at Membrane Contacts: Dynamics and Functions Beyond Lipid Homeostasis.
    Jiesi Xu, Xun Huang.
      Membrane contact sites (MCSs), regions where the membranes of two organelles are closely apposed, play critical roles in inter-organelle communication, such as lipid trafficking, intracellular signaling, and organelle biogenesis and division. First identified as "fraction X" in the early 90s, MCSs are now widely recognized to facilitate local lipid synthesis and inter-organelle lipid transfer, which are important for maintaining cellular lipid homeostasis. In this review, we discuss lipid metabolism and related cellular and physiological functions in MCSs. We start with the characteristics of lipid synthesis and breakdown at MCSs. Then we focus on proteins involved in lipid synthesis and turnover at these sites. Lastly, we summarize the cellular function of lipid metabolism at MCSs beyond mere lipid homeostasis, including the physiological meaning and relevance of MCSs regarding systemic lipid metabolism. This article is part of an article collection entitled: Coupling and Uncoupling: Dynamic Control of Membrane Contacts.
    Keywords:  lipid biosynthesis; lipid composition; lipid degradation; lipid functions; membrane contact site
    DOI:  https://doi.org/10.3389/fcell.2020.615856
  38. Stem Cell Res Ther. 2021 Jan 13. 12(1): 59
    Novel pharmacotherapy: NNI-362, an allosteric p70S6 kinase stimulator, reverses cognitive and neural regenerative deficits in models of aging and disease.
    Nathalie Sumien, Matthew S Wells, Akram Sidhu, Jessica M Wong, Michael J Forster, Qiao-Xi Zheng, Judith A Kelleher-Andersson.
      Aging is known to slow the neurogenic capacity of the hippocampus, one of only two mammalian adult neurogenic niches. The reduction of adult-born neurons with age may initiate cognitive decline progression which is exacerbated in chronic neurodegenerative disorders, e.g., Alzheimer's disease (AD). With physiologic neurogenesis diminished, but still viable in aging, non-invasive therapeutic modulation of this neuron regeneration process remains possible. The discovery of truly novel neuron regenerative therapies could be identified through phenotypic screening of small molecules that promote adult-born neurons from human neural progenitor cells (hNPCs). By identifying neuron-generating therapeutics and potentially novel mechanism of actions, therapeutic benefit could be confirmed through in vivo proof-of-concept studies. The key aging and longevity mTOR/p70S6 kinase axis, a commonly targeted pathway, is substrate for potential selective kinase modulators to promote new hippocampal neurons from NPCs. The highly regulated downstream substrate of mTOR, p70S6 kinase, directly controls pleiotropic cellular activities, including translation and cell growth. Stimulating this kinase, selectively in an adult neurogenic niche, should promote NPC proliferation, and cell growth and survival in the hippocampus. Studies of kinase profiling and immunocytochemistry of human progenitor neurogenesis suggest that the novel small molecule NNI-362 stimulates p70S6 kinase phosphorylation, which, in turn, promotes proliferation and differentiation of NPCs to neurons. NNI-362 promoted the associative reversal of age- and disease-related cognitive deficits in aged mice and Down syndrome-modeled mice. This oral, allosteric modulator may ultimately be beneficial for age-related neurodegenerative disorders involving hippocampal-dependent cognitive impairment, specifically AD, by promoting endogenous hippocampal regeneration.
    Keywords:  Allosteric modulator; Alzheimer’s disease; BrdU+ neuron survival; Hippocampal neuron regeneration; Human neural progenitors; mTOR/p70S6 kinase axis
    DOI:  https://doi.org/10.1186/s13287-020-02126-3
  39. Cells. 2021 Jan 12. pii: E138. [Epub ahead of print]10(1):
    Identification of Cathepsin D as a Plasma Biomarker for Alzheimer's Disease.
    Jae-Whan Kim, Soon-Young Jung, Youngbin Kim, Hansol Heo, Chang-Hyung Hong, Sang-Won Seo, Seong-Hye Choi, Sang-Joon Son, Seongju Lee, Jaerak Chang.
      Although Alzheimer's disease (AD) is the most common neurodegenerative disease, there are still no drugs available to treat or prevent AD effectively. Here, we examined changes in levels of selected proteins implicated in the pathogenesis of AD using plasma samples of control subjects and patients with cognition impairment. To precisely categorize the disease, fifty-six participants were examined with clinical cognitive tests, amyloid positron emission tomography (PET) scan, and white matter hyperintensities scored by magnetic resonance imaging. Plasma cathepsin D levels of the subjects were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Correlation of plasma cathepsin D levels with AD-related factors and clinical characteristics were examined by statistical analysis. By analyzing quantitative immunoblot and ELISA, we found that the plasma level of cathepsin D, a major lysosomal protease, was decreased in the group with amyloid plaque deposition at the brain compared to the control group. The level of plasma cathepsin D was negatively correlated with clinical dementia rating scale sum of boxes (CDR-SB) scores. In addition, our integrated multivariable logistic regression model suggests the high performance of plasma cathepsin D level for discriminating AD from non-AD. These results suggest that the plasma cathepsin D level could be developed as a diagnostic biomarker candidate for AD.
    Keywords:  Alzheimer’s disease; CDR-SB score; cathepsin D; plasma biomarker
    DOI:  https://doi.org/10.3390/cells10010138
  40. Front Cell Neurosci. 2020 ;14 619712
    AAV-Mediated GALC Gene Therapy Rescues Alpha-Synucleinopathy in the Spinal Cord of a Leukodystrophic Lysosomal Storage Disease Mouse Model.
    Michael S Marshall, Yazan Issa, Gregory Heller, Duc Nguyen, Ernesto R Bongarzone.
      Krabbe's disease (KD) is primarily a demyelinating disorder, but recent studies have identified the presence of neuronal protein aggregates in the brain, at least partially composed by alpha-synuclein (α-syn). The role of this protein aggregation in the pathogenesis of KD is largely unknown, but it has added KD to a growing list of lysosomal storage diseases that can be also be considered as proteinopathies. While the presence of these protein aggregates within the KD brain is now appreciated, the remainder of the central nervous system (CNS) remains uncharacterized. This study is the first to report the presence of thioflavin-S reactive inclusions throughout the spinal cord of both murine and human spinal tissue. Stereological analysis revealed the temporal and spatial accumulation of these inclusions within the neurons of the ventral spinal cord vs. those located in the dorsal cord. This study also confirmed that these thio-S positive accumulations are present within neuronal populations and are made up at least in part by α-syn in both the twitcher mouse and cord autopsied material from affected human patients. Significantly, neonatal gene therapy for galactosylceramidase, a treatment that strongly improves the survival and health of KD mice, but not bone marrow transplantation prevents the formation of these inclusions in spinal neurons. These results expand the understanding of α-syn protein aggregation within the CNS of individuals afflicted with KD and underlines the tractability of this problem via early gene therapy, with potential impact to other synucleinopathies such as PD.
    Keywords:  Parkinson's disease; alpha-synuclein; globoid cell leukodystrophy; proteinopathies; spinal cord
    DOI:  https://doi.org/10.3389/fncel.2020.619712
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